Phenotype of spl36 lesion mimic
Under normal planting conditions in summer, the leaves of spl36 did not change significantly from those of the wild type (WT) before the tillering stage. At tillering stage, the lesion mimic appeared in the leaf apex (Fig. 1A). From the tillering stage to the heading stage, these necrotic spots became more severe and gradually spread to the whole leaf (Fig. 1B). To investigate whether spl36 is induced by light like most lesion mimics, mutant spl36 leaves were covered with 2–3 cm aluminum foil at the tillering stage, with the uncovered mutant leaves used as additional controls. After even days, it was observed that no spread of lesion mimics had occurred in the covered area of the covered leaves, while the lesion mimics on the uncovered control leaves (Fig. 1C). This shows that the lesion mimic phenotype arising from mutant spl36 is induced by light. Meanwhile, the main agronomic traits of mutant spl36 such as plant height, grain number per panicle, and 1000-grain weight were significantly reduced (Fig. 1D–I).
SPL36 Gene Regulates Plant Growth and Development
Because of the negative agronomic changes in mutant spl36, and chloroplasts are the main site of photosynthesis. We speculated that the growth and development of the plants were affected after the appearance of the mutant lesion mimic phenotype (Han et al., 2015). We used a transmission electron microscope to observe chloroplast ultrastructure and found that the chloroplasts of mutant spl36 were atrophied and the volume of chloroplasts became smaller, along with disorganized lamellae inside the chloroplasts (Fig. 2A–D).We speculated that the growth and development of the plants were affected after the appearance of the mutant lesion mimic phenotype. Measurement of the chlorophyll content of wild-type Yundao and mutant spl36 at the tiller peak revealed that both chlorophyll a and chlorophyll b of mutant spl36 were significantly reduced compared with the wild-type (Fig. 2E). We further measured the photosynthetic rate of the plants during this period, and the results showed that the net photosynthetic rate of the mutants was significantly reduced (Fig. 2F). Therefore, the SPL36 gene regulates plant growth and development through changes in chloroplast structure.
SPL36 Regulates ROS Accumulation and Cell Death in Rice
The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay is designed to detect DNA fragmentation, which is a marker of programmed cell death (Kim et al., 2009). The TUNEL signal in the nuclei of mutant spl36 cells was intense and randomly distributed, whereas only a weak TUNEL signal was detected in the wild type (Fig. 3A–D). In addition, the accumulation of reactive oxygen species (ROS) at high concentrations leads to an oxidative burst, which causes cell damage and even triggers programmed cell death (Kim et al., 2010). The content of H2O2 and the activity of peroxidase (POD) are directly related to the accumulation of ROS. Superoxide dismutase (SOD) plays an important role in scavenging O2− in plants. Through the detection of H2O2 content, POD activity and SOD activity, it was found that a large amount of H2O2 accumulated in the mutant spl36 (Fig. 3E), while the activities of POD and SOD in the mutant spl36 were significantly reduced (Fig. 3G–H). This decrease in enzyme activity would negatively affect the removal of the related peroxide and negative oxygen ions, resulting in the accumulation of ROS. In addition, membrane lipid peroxidation occurs when plant organs age or suffer damage under stress. Malondialdehyde (MDA) is the final decomposition product of membrane lipid peroxidation, and the content of MDA can reflect the degree of damage in a stressed plant. We found that the MDA content was significantly higher in the mutant spl36 than in the wild-type (Fig. 3F). These results indicate that the lesion mimics in spl36 mutants are caused by ROS accumulation and irreversible membrane damage. In addition, loss of SPL36 function triggers the PCD pathway, ultimately leading to the appearance of the spl36 lesion mimic phenotype.
SPL36 Regulates Defense Responses in Rice
It has been reported that most rice lesion mimic mutants have enhanced resistance to pathogens. To investigate whether the resistance of mutant spl36 to rice pathogens was enhanced, we performed an inoculation assay on wild-type Yundao and mutant spl36 at the tillering stage, and used the leaf clipping method to plant the rice bacterial blight strain HM73. Changes in the inoculation site and the length of the lesion mimics were observed at 5 and 10 days after inoculation, respectively. We found that the leaf apex of the wild-type showed obvious necrotic spots at five days after inoculation, while the mutant did not show obvious disease spots; the length of the wild-type disease spots was significantly longer than that of the mutant 10 days after inoculation (Fig. 4A–E). This shows that the resistance to bacterial pathogens is significantly enhanced after the emergence of the mutant spl36 disease spots. To further explore the mechanism of enhanced resistance of mutant spl36 to bacterial pathogens, we examined the expression of defense-related genes in wild-type and mutants at the tillering stage by using qRT-PCR, and the results showed that the expression levels of defense genes MAPK12, WRKY53, BIMK2, AOS2, ASP90, LYP6, PR2, PR1a, and PR1b were significantly elevated (Fig. 4F). Thus, loss of SPL36-encoded protein function triggers a rice defense response, which leads to enhanced resistance of mutant spl36 to pathogens.
Genetic Analysis and Map-Based Cloning of SPL36 Gene
Mutant spl36 was used as the female parent to be hybridized with ZF802 of the Japonica cultivar TN1. The F1 plants did not show the phenotype of lesion mimic, and the segregation ratio of normal phenotype and lesion mimic phenotype in the F2 population was essentially in compliance with the 3: 1 ratio, indicating that the spl36 phenotype is caused by mutations in a single recessive nuclear gene (Supplementary Table 1). A selection of polymorphic markers from 238 insertion and deletion tags mapped 21 F2 individuals with a lesion mimic phenotype, and we mapped the mutation site to a location between chromosome 12 B12-5 and B12-6 (Fig. 5A). The SPL36 location was further refined to a location between JHL-3 and JHL-7 by genotyping 148 mutant F2 individuals from the same cross and adding four additional polymorphic tags (Fig. 5B). Using an additional 554 F2 mutant individuals and four newly developed polymorphic tags, we finally mapped SPL36 to a 60 kb region between markers InDel1 and InDel2 (Fig. 5C). Website inquiry (http://rice.plantbiology.msu.edu/) predicted that the region had 11 open reading frames (ORFs), which included seven expressed proteins and four functional proteins (Fig. 5D). Through sequencing and alignment, we found that the gene LOC_Os12g08180 was mutated (Fig. 5E), and nucleotide C at position 1462 in the coding region of this gene was replaced with T (Fig. 5F), resulting in the change of the encoded amino acid from arginine to cysteine (Fig. 5G), so LOC_Os12g08180 was used as a candidate gene for SPL36.
Functional complementation of the spl36 mutant with LOC_Os12g08180
To verify whether the single base substitution in LOC_Os12g08180 was associated with the spl36 phenotype, we constructed the vector pGSPL36, which contained genomic DNA fragments including the promoter of the SPL36 gene in wild-type Yundao, and then introduced it into spl36 by Agrobacterium tumefaciens-mediated transformation. The corresponding empty vector pEmV was also transformed as a control. Of the 60 T0 plants which had been transformed, 54 were positive transformants, all of which showed the same normal phenotype as the wild-type (Fig. 6A), while the plants transformed with the control vector showed the same lesion mimic phenotype as the mutant spl36 (Fig. 6B), demonstrating that LOC_Os12g08180 was SPL36, and that the single base substitution in spl36 led to the appearance of the lesion mimic phenotype of the plants.
Expression pattern analysis of SPL36
We used real-time quantitative PCR (qRT-PCR) to analyze the expression of SPL36 in various organs. The results showed that SPL36 was expressed in the organs, with higher expression in leaves, leaf sheaths, and roots and lower expression in stems and panicles. SPL36 expression was significantly higher in all organs of mutant spl36 compared to the wild-type Yundao (Fig. 7A). To analyze the spatiotemporal expression pattern of SPL36 more precisely we constructed the vector pSPL36: GUS by fusing the GUS gene with the promoter of SPL36 in the wild-type. We also utilized Agrobacterium tumefaciens-mediated transformation to obtain transgenic plants. We stained various organs of the transgenic positive plants and observed GUS signal maps in various tissues (Fig. 7B–F), which was consistent with the qRT-PCR results. These results suggested that SPL36 was expressed in all organs and at all developmental stages.
Subcellular Localization of SPL36 Protein
To determine the subcellular localization of SPL36, the full-length coding sequence of SPL36 was fused to the N-terminus of green fluorescent protein (GFP). When transiently expressed in rice protoplasts, the GFP signal appeared on the plasma membrane (Fig. 8A–D). To verify this observation, we transformed the plasmid containing the SPL36-GFP fusion vector into Nicotiana benthamiana leaves, resulting in the detection of the SPL36-GFP protein on the membrane (Fig. 8E–H). These results show that the SPL36 protein localizes to the membrane.
SPL36 is involved in salt stress-responsive responses in rice
After verifying that the single base substitution of LOC_Os12g08180 was responsible for the lesion mimic phenotype of mutant spl36, we found that this gene encodes a receptor-like protein kinase 2 precursor. Plant receptor-like protein kinases play an important role in the process of plant signal transduction and are indispensable carriers which can perceive the signals of growth and development and external environmental stresses by phosphorylation of functional proteins resulting in conformational changes (Lally et al., 2001); plant receptor-like protein kinases also play a regulatory role in plant growth and development and disease resistance defense responses (Afzal et al., 2008; Li Liyun et al., 2008) and most receptor-like protein kinases are related to stress responses. To investigate whether SPL36 is involved in stress response-related pathways, we performed a salt stress assay in flat dishes and hydroponic seedlings for the wild-type and mutant In the plate assay, in the absence of salt treatment, we found no significant difference in the germination rate of mutant spl36 and wild-type over a one-week period. In the case of salt treatment, the germination rate of both mutant and wild-type decreased significantly, while the germination rate of the wild-type was also significantly lower than that of the mutant. At day 9 of germination we counted the length of the supra-root portion of the salt-treated and control seedlings, and there was no significant difference in the length of the supra-root portion between wild-type and mutant in the control group while the length of the supra-root portion of the mutant was significantly lower than that of wild-type in the case of salt treatment (Supplementary Fig. 1). In addition, we also treated the wild-type and mutant seedlings hydroponically for four weeks with salt, returning them to normal conditions after three days of treatment. The results showed insignificant changes in the wild-type after three days of treatment with the phenotype recovering after restoration of normal conditions, while the mutant spl36 showed significant leaf bending after the salt treatment while the phenotype did not recover or even died after restoring normal conditions. Our statistical analysis of fresh weight, conductivity as well as final survival of plants before and after treatment as well as controls revealed that mutant spl36 was more sensitive to salt treatment (Fig. 9). In summary, SPL36 is involved in salt stress-responsive responses in rice.